The majority of retaining α-glycosidases are believed to adopt the classical double displacement mechanism to catalyze their reactions, which features a catalytic nucleophilic residue, a general acid/base residue, two oxocarbenium-ion like transition states and one covalent glycosyl-enzyme intermediate. In my thesis, the catalytic mechanisms of three retaining alpha-glycosidases were investigated in detail as follows. HPA is an enzyme which is responsible for hydrolyzing starch into shorter

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... osaccharides. Several 2-deoxy-2,2-dihalo maltosyl chlorides were synthesized and tested as potential mechanism-based inhibitors of HPA, in the hope of trapping its covalent glycosyl-enzyme intermediate for crystallographic studies. Unfortunately, none of newly-synthesized compounds could cause time-dependent inactivation of HPA. By employing our newly developed in situ elongation strategy, 5-fluoro-α-D-glucopyranosyl fluoride and 5-fluoro-β-L-idopyranosyl fluoride showed kinetic behavior consistent with the proposed in situ elongation-inactivation process, allowing the trapping and further kinetic and structural analysis of the covalent intermediate of HPA. These structures provide interesting mechanistic insights into the catalytic mechanism of HPA. TreS is an enzyme which catalyzes the reversible interconversion of maltose and trehalose. 5-Fluoro glycosyl fluorides were shown to be mechanism-based inhibitors of this enzyme by accumulating the covalent glycosyl-enzyme intermediate. The trapped intermediate was subjected to protease digestion followed by MS analysis of the resultant peptides to identify the catalytic nucleophile residue as D230. The inability of TreS to carry out transglycosylation reactions onto exogenously added acceptors establishes the intramolecular nature of the rearrangement reaction, consistent with previous studies on other TreS enzymes. All studies support a double displacement mechanism involving an intramolecular "glucose flipping" step as the catalytic mechanism of this enzyme. SpGH101 i [...]